Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

Antimicrobial resistance and species identification of staphylococci isolated from the meat of wild rabbits (<Emphasis Type="Italic">Oryctolagus cuniculus</Emphasis>) in Slovakia

In 2009, a total of 113 strains of staphylococci were isolated from the thigh muscles of ten hunted and 20 farmed wild rabbits (Oryctolagus cuniculus) in the Slovak Republic. Only two isolates (1.8%) possessed coagulase activity, the rest of 111 staphylococcal isolates were coagulase-negative. Among them, six isolates (5.4%) showed the production of DNase. In each isolate, resistance to eight antibiotics by means of agar dilution test was tested. Based on these results, 110 isolates were found to be resistant to at least one antibiotic. Only one isolate was susceptible to all eight antibiotics tested. Another two isolates were susceptible, however, they showed intermediate susceptibility to cefoxitin. Resistance to ampicillin (78.8%), erythromycin (58.4%), penicillin (51.3%) and oxacillin (46.0%) was found most frequently. Twenty-six isolates (23.0%) were resistant to novobiocin. On the other hand, resistance to cefoxitin (8.0%) and gentamicin (1.8%) were quite rare. Fifteen percent of isolates were resistant to one antibiotic, simultaneous resistance to two, three, four and five antibiotics was confirmed in 22.1%, 23.9%, 21.2% and 13.3% of isolates, respectively. Except for two coagulase-positive Staphylococcus aureus isolates (1.8%), seven species of coagulase-negative staphylococci were identified using the MALDI BioTyper (TM) sytem as follows: Staphylococcus warneri (45.1%), Staphylococcus epidermidis (21.2%), Staphylococcus pasteuri (13.3%), Staphylococcus xylosus (8.0%), Staphylococcus capitis (7.1%), Staphylococcus haemolyticus (1.8%) and Staphylococcus cohnii ssp cohnii (1.8%).     

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Abstract Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H₂ turnover per gram soil. Dilution did not affect the fraction of H₂/CO₂-dependent methanogenesis which made up 22% of total CH₄ production. However, dilution resulted in a ten fold decrease of the H₂ steady state partial pressure from approximately 4 to 0.4 Pa indicating that H₂/CO₂-dependent methanogenesis was more or less independent of the H₂ pool. The rates of H₂ production calculated from the H₂ turnover rate constants and the H₂ steady state partial pressures accounted for only < 5% of H₂/CO₂-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H₂/CO₂-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H₂ transfer within syntrophic bacterial associations and was not significantly affected by the outside H₂ pool.     

Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology

Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH2A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation.     

Vectors for Expression of Signal Peptide-Dependent Proteins in Baculovirus/Insect Cell Systems and Their Application to Expression and Purification of the High-Affinity Immunoglobulin Gamma Fc Receptor I in Complex with Its Gamma Chain

Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER). The vectors code for N-terminal and C-terminal affinity tags including 3 × FLAG and Twin-Strep, which represent tags compatible with efficient translocation to the ER as well as with purification under mild conditions that preserve protein structure and function. As a model, we used our system to express and purify the engineered high-affinity immunoglobulin gamma Fc receptor I (CD64) in complex with its gamma subunit (γ-chain). We demonstrate that CD64 expressed in complex with the γ-chain is functional in immunoglobulin G (IgG) binding. The sedimentation of CD64 in complex with IgG suggests individual CD64/IgG complexes in addition to formation of high-molecular weight complexes. In summary, our vectors can be used as a tool for expression of membrane proteins, other secretory pathway proteins and their protein complexes.     

Enrichment of Murine CD68+CCR2+ and CD68+CD206+ Lung Macrophages in Acute Pancreatitis-Associated Acute Lung Injury

Acute lung injury (ALI) is an important cause of mortality in critically ill patients. Acute pancreatitis (AP) is one of the risk factors for developing this syndrome. Among the inflammatory cells, macrophages have a key role in determining the severity of the acute lung injury. In the lungs, macrophages constitute a heterogeneous cell population distributed in different compartments. Changes in not only the macrophage count, but also in their phenotype have been seen during the course of lung injury. A murine ductal ligation model of acute pancreatitis showed substantial morphological changes in the pancreas and lungs. Immunohistochemistry showed neutrophil recruitment into both organs after 9 hours and later on. F4/80⁺ cells in the pancreas increased in the ligated animals, though there was not a significant difference in their number in the lungs as compared to sham operated animals. Flow cytometry analysis of lung macrophages demonstrated an enrichment of F4/80⁻ CD68⁺CCR2⁺ and F4/80⁻ CD68⁺CD206⁺ lung macrophages in ligated animals (AP) as compared to the sham operated group. The level of interleukin-6 in plasma increased 3 hours after ligation compared to the sham operated group, as a first indicator of a systemic inflammatory response.This study suggests a role for F4/80⁻ CD68⁺ macrophages in the pathogenesis of acute lung injury in acute pancreatitis. Studying lung macrophages for different phenotypic markers, their polarization, activation and recruitment, in the context of acute lung injury, is a novel area to potentially identify interventions which may improve the outcome of acute lung injury.     

How functional is functional? Ecological groupings in terrestrial animal ecology: towards an animal functional type approach

Understanding mechanisms to predict changes in plant and animal communities is a key challenge in ecology. The need to transfer knowledge gained from single species to a more generalized approach has led to the development of categorization systems where species’ similarities in life strategies and traits are classified into ecological groups (EGs) like functional groups/types or guilds. While approaches in plant ecology undergo a steady improvement and refinement of methodologies, progression in animal ecology is lagging behind. With this review, we aim to initiate a further development of functional classification systems in animal ecology, comparable to recent developments in plant ecology. We here (i) give an overview of terms and definitions of EGs in animal ecology, (ii) discuss existing classification systems, methods and application areas of EGs (focusing on terrestrial vertebrates), and (iii) provide a “roadmap towards an animal functional type approach” for improving the application of EGs and classifications in animal ecology. We found that an animal functional type approach requires: (i) the identification of core traits describing species’ dependency on their habitat and life history traits, (ii) an optimization of trait selection by clustering traits into hierarchies, (iii) the assessment of “soft traits” as substitute for hardly measurable traits, e.g. body size for dispersal ability, and (iv) testing of delineated groups for validation including experiments.     

The association between estrogen receptor alpha polymorphisms and the risk of prostate cancer in Slovak population

The aim of our study was to evaluate the effect of two polymorphisms in the estrogen receptor alpha, PvuII and XbaI, on the development of prostate cancer within Slovak population, as well as their correlation with selected clinical characteristics. The study was performed using 311 prostate cancer patients and 256 healthy male controls. Both polymorphisms were significantly associated with higher risk of prostate cancer development. At the same time, the CC genotype of PvuII polymorphism (OR = 1.98; 95 % CI 0.94–4.21; p = 0.05) and the AG genotype of XbaI polymorphism (OR = 1.74; 95 % CI 1.0–3.02; p = 0.04) significantly contributed to the development of low-grade carcinoma, while the AG and GG genotypes of the XbaI polymorphism contributed mainly to the development of high-grade prostate cancer (OR = 1.83; 95 % CI 1.12–3.01; p = 0.01 and OR = 2.13; 95 % CI 1.06–4.19; p = 0.03, respectively). Similarly, the AG and GG genotypes of XbaI polymorphism showed significant association with prostate cancer in patients with serum PSA level ≥10 ng/ml. Both polymorphisms were found at the same time to be more frequent in patients diagnosed before the age of 60. We conclude on the basis of these results that PvuII and XbaI polymorphisms of estrogen receptor alpha might be associated with prostate cancer risk within Slovak population. Although this is a pilot study and, as such, more detailed investigations are needed to confirm the role of these polymorphisms in prostate cancer development and progression within said Slovak population, our results might still provide a valuable basis for further research with larger patient groups.